1Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, 4010 Debrecen, Egyetem ter 1, Life Science Building, 4010 Debrecen, Hungary2Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, 4010 Debrecen, Hungary

Abstract

Dendritic cells (DCs) can regulate all elements of the immune system, and therefore are an ideal target for vaccination. During the last two decades, as a result of extensive research, DCs became the primary target of antitumor vaccination as well. A critical issue of antitumor vaccination is the phenotype of the dendritic cell used. It has been recently shown that several nuclear hormone receptors, and amongst them the lipid-activated nuclear receptor and peroxisome proliferator-activated receptor gamma (PPAR), have important roles in effecting the immunophenotype of human dendritic cells. It regulates primarily lipid metabolism and via this it influences the immunophenotype of DCs by altering lipid antigen uptake, presentation, and also other immune functions. In this review, we summarize the principles of antitumor vaccination strategies and present our hypothesis on how PPAR-regulated processes might be involved and could be exploited in the design of vaccination strategies.

1. Dendritic Cells in Tumors

Dendritic cells (DCs) were discovered in mouse spleen by Ralph Steinman and
Zanvil Cohn in 1973 [1].
Immature dendritic cells (IDCs) are sentinels of the immune system, continuously monitoring peripheral tissues for invaders, capture and process antigens, and migrate to the draining lymph nodes where they present peptides to naive Treg cells (T cells) and
activate them [2]. The full activation of T cells
requires special peptide-MHCI
or peptide-MHCII complexes and additional signals from DCs in the form of various costimulatory
molecules and cytokines.
Furthermore, activated CD4+ T cells could be polarized to T helper 1
(Th1) and T helper 2 (Th2) subtypes. These processes are dependent on the
cytokines interleukin-12
(IL-12), IL-4, and IL-10 secreted by mature DCs (MDCs). In response to IL-12, T
cells polarize to Th1 and enhance CD8+ cytotoxic T-cell response
against tumor cells or pathogens, while IL-4- and IL-10-activated Th2 cells
promote humoral immune response and/or tolerance. Another important point is
that DCs can induce T-cell tolerance to self-antigens and via this prevent and
reduce autoimmune diseases.

Importantly, it appears that tumor tissues have
characteristic immune environments with distinct DC subset distributions.
Different DC subset localization within the compartments of tumor has been reported in colorectal cancer and oral squamous cell carcinoma patients. Dadabayev et al. investigated the
infiltration pattern of DCs in human colorectal tumor samples analyzed with S100 and HLA class II DC markers. S100+ and CD1a+ DCs were found in tumor epithelium, in parallel with intraepithelial CD4+ or CD8+ T-cell infiltration and suggested increased disease-free survival, while HLA class II+ cells were observed
in the stromal compartment, correlated with adversed outcome of the tumor
[3]. Later they utilized CD208 (DC-LAMP)
marker for marking MDCs and proved that CD208+ DCs were detectable in the peritumoral area, and infiltration of MDCs into tumor epithelium was correlated also with decreased patient survival
[4]. In primary squamous cell carcinoma
patients, IDCs and MDCs were characterized with distinct tissue localization
patterns. Immature Langerhans
cells (LCs) and DC-SIGN+ interstitial DCs were found inside the tumor tissue while the number of mature CD208+ DCs was limited. Moreover, CD123+ plasmacytoid DC representation in the tumor area was correlated with poor survival
[5]. Importantly, DCs interact with tumor cells and cytokines produced by tumor cells or immune cells influence DC function and
maturation. The tumor microenvironment affects DC differentiation from CD14+ monocytes and haematopoietic precursors promoting an early and dysfunctional
maturation of DCs. Several reports described reduced the number of
DC in peripherial blood, tumor tissues, and draining lymph nodes in cancer patients. Partial maturation of DCs by tumor-derived factors like IL-10, vascular endothelial growth factor (VEGF), and TGF- induces self-tolarence and promotes conversion of naive T cells to regulator T cells,
favoring development of suppressive T cells. Presence of tolerogenic T cells in tumor beds induces local immune suppression and alters the function of anticancer effector T cells. These cells, isolated from draining lymph nodes of patients with pancreas or
breast cancer secrete IL-10 and TGF-, prevent activated CD4+ CD25- and CD8+ effector T cells, and suppress tumor-specific immune response [6]. Apart from the fact that DCs are involved in activation of Treg, there is an increasing amount of evidence about T cells, which can be recruited into tumors and affect DC development. Decreased CD80 and CD86 cell surface markers by T cells lead to reduced T-cell stimulatory ability of DCs
[7, 8]. Immunosuppressive B7-3/4 molecules are upregulated on DCs upon DC-Treg interaction reserving a possible
feedback loop to generate more regulatory T cells
[9, 10]. Another immunosuppressive cycle is the conversion of DCs by Treg-secreted INF- and CTLA-4 into an indolamine 2, 3-dioxigenase
(IDO) expressing cells which induce Treg generation and effector T-cell
apoptosis [11, 12].

Classically, CD4+ T cells have been categorized into Th1, Th2,
Treg, and Th17 subsets. However,
TGF- has a crucial role in Treg and Th17 cell
development, the dichotomy of Treg/Th17 is dependent on IL6. Only a few pieces of evidence has been
reported on the presence and regulation of Th17 cells by IL-2 in human cancer
and experimental tumors. Muranski et al. reported that tumor-specific Th17 polarized cells mediated successful treatment of large established tumor in cutaneous melanoma-bearing mice. The therapeutic effect of the cells was dependent on their INF production [13].

Modulating factors released by the tumor environment cause defective functional maturation of DCs and affect the differentiation of immature myeloid-derived suppressing cells (MDSCs). The portion of MDSC is significantly increased in spleen, peripheral
blood, and bone marrow of tumor-bearing patients and correlates with tumor progression [14–16].

In conclusion, DCs are one of the potent regulator cells in tumor development.
The effects of DCs in cancer patients are contraversial: several reports demonstrated that myeloid-derived MDCs induce effective antitumor
immune response and tumor regression. In spite of this, the suppressive tumor environment can alter the properties of DCs. The functional defects of DCs have an essentional role in cancer
patient to impede succesful antitumor immune response. These tolerogenic DCs function
as tumor-promoting cells. The future challenge of anticancer-based therapies is
to overcome DC tolerogenecity and to reduce their negative effects in tumor progression.

2. Dendritic Cell-Based Cancer Therapy

It is clear that however in low number DCs are present in
tumors and can be used to elicit antitumor immune response. The challenge and goal of anticancer therapy is to elicit an effective cellular immune response against tumor cells and evoke clinical response in treated patients with negligible side effects. The
discovery of isolation techniques and methods for differentiating DCs in vitro gave us the possibility to generate DCs that could be loaded by tumor-specific antigens or peptides. In this therapeutic approach, one can define DC-vaccine
as a DC loaded with tumor-specific antigen. The first DC-based clinical trial against B-cell lymphoma
was reported by Hsu et al. [17]. One important question in DC vaccination is to decide whether to use an ex vivo or in vivo vaccination strategy. The ex vivoapproaches (see Figure 1) allow to monitor the quality of the cells during the differentiation procedure, analyze cell surface markers, the proper maturation state, cell viability, or subtype specificity of DCs by FACS analysis. It is also possible
to evaluate the effective tumor antigen-specific T-cell response by ELISpot, mixed leukocyte reaction (MLR) before targeted DCs are introduced back to the patient. The
possible sources of human DCs are CD34+ precursors, hematopoetic progenitors, and monocytes, isolated from blood by cytopheresis, adherent techniques or magnetic-based immunoselection, or immunodepletion
[18–20]. IDCs can be differentiated
from peripheral blood-derived monocytes in vitro in the
presence of GM-CSF and IL-4 [18, 20]. Alternatively, DC
precursors can be isolated from human peripheral blood, but for effective
anticancer therapy one has to obtain high amount of targetable DCs. In a
clinical trial, FLT-3 ligand-expanded DCs were prepared from the blood of colon
and nonsmall cells of lung cancer patients. Because of the
limited blood DC number, patients underwent FLT-3 treatment before DC
isolation. As a result, three times more PBMC was
obtained from these patients after standardized leukopheresis as compared to
control patients. The
isolated patient-derived DCs showed immature CD83-/// phenotype, but after two days
in culture, cells started to express CD83, elevated levels of CD86 and CCR7 proteins, which
reflect MDC phenotype and migration capacity [21]. DC-vaccine studies utilize DCs loaded with peptide fragments or whole proteins providing an opportunity to present all potential peptide sequences of the antigen to recognize even
more specific T-cell clones and tumor lysates exogenously [22]. Alternatively, one can target DCs endogenously with antigen-coded mRNA or cDNA
[23]. After loading IDCs with
tumor-specific antigens, it is very important to add adequate maturation agents
(e.g., proinflammatory cytokines, LPS, CD40L) to ensure that DCs achieve their maximum migratory capacity to the lymph nodes, otherwise only a small portion of antigen-loaded DCs
could migrate to the site of the naive T-cell activation. Following quality control
steps, the generated DC vaccines have to be reinjected into the
patients. An important issue is also the DC injection
site. DC vaccines could be reinjected to patients by intravenous, subcutaneous,
intradermal, or intralymphatic injections.
In a clinical study, the efficiency of different injection sites was compared by Fong et al. [24]. According to their results,
the intradermal or intralymphatic administration was more effective compared to
intravenous injection. In general,
DC delivery via the skin is preferable to intravenous injection. Combining the
different routes of reinjections may
be beneficial, depending on the tumor localization. Most of the early phase I
clinical trials, using the ex vivo approach, have not shown long-term tumor regression or improved survival. Probably it is mostly due to the fact that in these
studies the researchers selected only advanced-stage cancer patients who were
immonosuppressed by recurrent tumors or by chemotherapy. However, an in vitro approach could provoke Th1 cell response in metastatic malignant melanoma [25].

Figure 1: General scheme of anticancer
vaccination. Dendritic cell progenitors (either
CD34+ or CD14+ cells) are obtained using cytopheresis.
Cells are differentiated using cytokines GM-CSF and IL-4. Immature dendritic cells are loaded with tumor lysate,
peptides, or expression vector. DC maturation is induced and DCs are reinjected
to patient.

As far as the migratory capacity of DCs is concerned, less than 5% of
the MDCs
reach the lymph nodes after intradermic injection [26]. Therefore, it would be more beneficial to activate and target DCs within the host. In this
case, we are not concerned with cell isolation or differentiation protocols, but rather DC-initiated tumor-specific immune responses have to be
monitored inside the body. Monoclonal antibodies and fusion construct can be
used for more productive tumor antigen delivery directly into the DCs and
probably the cells do not need to be cultured in vitro. Many vaccination studies target DC-specific c-type lectin receptors for efficient targeting of tumor antigens into the cells. These receptors bind to particular self- or nonself-sugar patterns by means of their carbohydrate
recognition domain (CRD) and have roles in endocytotic antigen uptake
[27]. One of them, DEC205/DC205, is expressed at high levels by MDC in mice, but human B cells, NK cells, monocytes, and macrophages also express this receptor [28]. Bozzacco et al. designed a
fusion monoclonal antibody construct by taking the light and heavy chain coding
cDNA sequence of an anti-DEC205 antibody and by inserting different gag p24
peptides at the carboxy terminus of the heavy chain. According to their results,
these antibodies increased antigen presentation in the treated HIV-infected
patients. They could further demonstrate DC-primed cross-presentation of
internalized, nonreplicating proteins to MHCI complexes inducing CD8+ T-cell activity [29]. These results support the feasibility of engineering
tumor-specific peptide fragment into DC-targeted antibodies against various
types of cancer in vivo.

3. The Role of the PPAR Receptor in Dendritic Cells

3.1. PPAR-Altered Phenotype of DCs

Nuclear hormone receptors are ligand-activated transcription factors. There are three different PPAR isoformes in the human body and these show distinct tissue-specific distribution with different
physiological functions. PPAR is most highly expressed in the liver, skeletal muscle, kidney, and heart, and it regulates fatty acid
oxidation [30]. PPARδ shows a ubiquiter distribution while PPAR expression can be detected in various cell
types like adipocytes, macrophages, and DCs. The receptor was initially described in mouse
adipose tissue [31]
and its role in myeloid development was shown by Nagy and Tontonoz in 1998 [32, 33]. PPAR knockout mice are lipodystrophic and die of placental defect, showing the essential regulatory role for the receptors in embryonic differentiation [34]. Moreover, high level of PPAR expression can be detected in monocyte-derived macrophages in atheroscleric lesions [33]. PPAR receptors
heterodimerize with retinoid X receptors (RXRs) in the nucleus and bind to certain receptor-specific response elements (PPREs) in the promoter or enhancer
regions of their target genes [35]. The PPRE contains direct
repeat sequences separated by one base pair (DR1). Endogenous or exogenous
ligands bind into the ligand-binding domain (LBD) of PPAR and modulate PPAR-mediated
gene expression. PPAR can be activated by components of the oxidized low-density lipoprotein (oxLDL) and prostaglandin derivate (e.g.,15d-PGJ2)
[32, 36]. Ligand activation of the
receptor induces the expression of CD36 scavenger receptor which in turn leads
to oxLDL uptake of macrophages and this metabolic process can lead to foam cell formation [32]. From these studies, we know that PPAR regulates fatty acid uptake into the cell by induced cell surface receptors and
it also promotes lipid storage and accumulation. Beside this fundamental regulatory role in metabolism, the receptor also functions as a
key modulatory factor in macrophage immune function [37]. Earlier microarray data suggested that the PPAR gene is upregulated during monocyte-to-DC
differentiation [38]. According to our experiments and those of
others, the receptor is immediately upregulated in cultured DCs, while it is barely detectable in monocytes [39]. We have shown that the
transcription factor in this system is active, because synthetic agonists induce dose-dependent gene
expression of the bone fide PPAR target gene FABP4 in IDCs
[39, 40]. Through global gene expression analysis, we found that PPAR-activated genes involved primarily in the first 6 hours are involved
primarly in lipid metabolism and transport (CD36, LXR, and PGAR). Genes,
coupled to the immune regulatory role of human DCs, were upregulated only for 24 and 120 hours
after ligand treatment. It is possible that immunophenotype of DCs could be altered by PPAR activation indirectly through activation of lipid metabolism and signaling
pathways [41].

In terms of DC-based vaccination therapy, the most important question is how PPAR activation might effect the DC-initiated immune responses and DC
phenotype. PPAR expression was first detected in murine DCs by
Faveeuw et al. and they reported that there is a PPAR receptor-dependent inhibition of IL-12
secretion of IDCs and MDCs [42]. Furthermore, it was also
shown that PPAR ligand activation caused anti-inflammatory
cytokine production in macrophages [37]. These
findings support the idea that PPAR might have an essential role in the APC-based
DC-vaccine therapies. The DC-secreted IL-12 is indispensable for Th1 cell promotion and CD8+ T-cell activation. Earlier publications by Gosett et al. and Nencioni et al. assessed that PPAR ligand activation alters the immunogenicity of
human monocyte-derived DCs [43, 44]. During DC maturation,
costimulatory molecules (CD40, CD80, and CD86) are upregulated on the surface of DCs [2]. Some bacterial products, such as LPS, are able to induce signals via TLR receptors or CD40 molecules induce IL-12 secretion of DCs.
They have also found that upon ligand activation of PPAR, the phenotype and cytokine expression patterns of the cells were changed [43]. PPAR ligands altered iDC-specific surface markers involved in APC function. The CD83 activation marker expression in treated MDCs was uneffected, which means that PPAR ligand-activated
cells showed mature phenotype. After ligand activation, elevated CD86 protein level was detected on the surface of MDCs. They also showed that activation of PPAR inhibits the secretion of IL-12p70 active form into the supernatant by MDCs while the levels of IL-6 and IL-10 were unchanged.
Furthermore, chemokines involved in Th1 cell recruitment (IP-10, RANTES, and
MIP1), were also decreased after ligand
treatment in the same study. Nencioni et al. later characterized the effects of PPAR on DC maturation and found that ligand activation reduced
the surface expression of CD1a molecule in a concentration-dependent manner, resulting in an unusual phenotype of
differentiated IDCs [44]. Lower levels of IL-10, IL-6, and TNF cytokines were measured upon ligand treatment.
PPAR agonist impaired the allogenic T-cell stimulating capacity in
MLR assays and the secreted INF concentration was also reduced. T-cell
activation capacity could not be restored by IL-12 administration suggesting
that the impaired T-cell activation of MDCs was not only due to lack of IL-12 expression but also
to other effects that modulate DC
maturation process were involved.

In conclusion, the PPAR ligand-activated
cells not only impede the
naive T cell to Th1 cell
differentiation, but these cells also showed decreased antigen-specific T-cell response. Appel et
al. reported that important anti-inflammatory effects of the receptor as ligand
activation of the PPAR receptors inhibited the LPS-activated MAP kinase and NF-B proinflammatory signaling pathways,
probably due to transrepression mechanism in DCs that subvert IL-12 expression [45].

Flow cytometry measurements performed by some of us largely
supported the phenotypic results reviewed above [39].
Furthermore, when treated DCs with
PPAR, we detected that enhanced endocytosis
in the form of enhanced latex bead uptake and ligand-treated cells were CD1a-. We could not detect any differences in case of
HLA-ABC molecule expression, suggesting that the MHCI-mediated
peptide antigen presentation capacity of the cell is probably not affected [46]. As reported by Angeli,
PPAR inhibits the expression of CCR7 on the surface of MDCs and this decreased the migration of
DCs in mice. In this model, TNF-induced epidermal LC motility from epidermis
to dermal lymph nodes was reduced by PPAR ligand treatment. They also found that ligand-activated PPAR impaired the steady-state migration of DCs
from the mucosal to the thoracic lymph nodes, but the maximal inhibitory effect
was detected at a considerably high
concentration of the PPAR agonist rosiglitazone (10 M) that suggested receptor-independent
effects [47].

Summarizing these data, PPAR activation in DCs prevented IL-12 secretion, lowered
CD80/CD86 ratio, and probably shifted naive T-cell
differentiation toward Th2 cells. According
to our own experiments, PPAR agonist
rosiglitazone at 2.5 M concentration did not decrease the activation of
allogeneic T cell and INF production [39]. So far, no one was
able to detect Th2 response in MLR in response to PPAR ligand activation.

3.2. The Role of PPAR in CD1d-Mediated Lipid Antigen Presentation

Szatmari et al. provided evidence that PPAR activation
could effect the lipid antigen presentation capacity of monocyte-derived DCs through
upregulated expression of CD1d molecule on the surface of DCs [39]. This finding links PPAR to invariant natural killer T (iNKT) cells. After
isolation, monocytes fail to express CD1 group I molecules (CD1a, b, c) but the CD1a protein is
upregulated during monocyte-to-IDC differentiation [20]. Inversely, the CD1 group II
molecule CD1d is expressed at high levels on
monocytes and downregulated on the surface
of DCs [39]. Induced signaling pathways are able to regulate
CD1d gene expression and lipid metabolism upon PPAR ligand treatment
[41]. Utilizing PPAR agonist treatment, Gogolak et al. could induce the expression of CD1d molecules along with downregulation of CD1a at both mRNA and protein levels [48]. Later we established
that PPAR ligand activation enhanced indirectly the CD1d expression by turning on endogenous
lipophilic ligand synthesis in the DCs through activation of the expression of retinol
dehydrogenase 10 (RDH10) and retinaldehyde dehydrogenase type 2 (RALDH2) enzymes, which are involved in retinol and retinal metabolism and endogenous all-trans retinoic acid (ATRA) production from retinol [46]. The intracellularly synthesized ATRA induced CD1d and other retinoic acid receptor alpha (RAR) target genes in DCs. We looked at the
functional role of PPAR-induced retinoid-regulated CD1d expression on
DC surface. DCs pulsed with synthetic alpha-galactosilceramide (GalCer) ligand for 24 hours elevated iNKT cell expansion and INF secretion [39, 41]. As CD1d-mediated lipid antigen presentation is essential for iNKT cell activation, we could conclude
that PPAR-induced CD1d expression can be translated to the increased
activation and proliferation
of iNKT cells under these in vitro conditions [39]. Our results suggest that combination of PPAR activator ligands along with GalCer during the differentiation of DCs might be beneficial in
iNKT-based adoptive transfer therapy (see Figure 2).

Figure 2: The molecular basis for the potential use of
PPAR-programed dendritic cells during tumor
vaccination.DC progenitors are differentiated
in the presence of PPAR agonists. A PPAR-programed DC showed increased CD1d
expression. In the presence of GalCer, the
treated DC is capable of
inducing iNKT cell expansion.
The adoptively transfered iNKTs can
induce activation of iDCs and IL-12 secretion in cancer patients. This can
lead to improved ability to kill tumor cells.

4. CD1d-Restricted iNKT Cells in Cancer Therapy

4.1. iNKT Cell-Based Anticancer Effects in Animal Models

Besides DCs and T cells, there are
other important cell types contributing to antitumor immunity. iNKT cells are a unique T-lymphocyte
population. These cells share both NK (CD161) and T-cell-specific markers (TCRs) on their surfaces.
iNKT cells have restricted T-cell receptors (TCRs): in mice, the most
frequently expressed -chain rearrangement is V14-J18 while human NKT cells express V24-J18/V11 TCRs (reviewed by Godfrey and Kronenberg [49]). For iNKT activation, it is essential to interact with
cells displaying the evolutionarily
conserved CD1d, nonclassical antigen-presenting molecules that present glicolipids in the context of hydrophobic antigen binding to these
cells [50, 51]. GalCer is the most frequently used lipid ligand for iNKT activation. It is derived from a marine sponge.

GalCer has shown antimetastatic activity in various experimental tumor models (e.g., B16 melanoma, Lewis lung carcinoma, FBL-3 erytroleukemia, Colon26, and RMA-S 3LL tumor cells) in vivo [52–54].
This effect of the compound was tested in CD1d-/-, Ja281-/- RAG-/-
NKT mice, which have no iNKT cells and in NK-depleted wild-type mice. The results indicated that the antitumor effect of
the glycolipid was abolished on all of the three tested genetic backgrounds in mice and the GalCer-mediated antimetastatic function likely acts
through iNKT cell activation and NK-like
effector function [52]. Adoptive transfer experiments
provided further proof for the key role of iNKT cell-secreted INF in the antimetastatic role of GalCer in mice. Furthermore, activation and proliferation of NK cells downstream to iNKT
activation, and subsequent INF production was also required to be essential for antimetastatic
cytotoxic activity in vitro and
in vivo [55, 56]. GalCer activates iNKT cells, which produce INF, and secondary activates NK cells. These activated NK cells have been implicated also in the regulation of angiogenesis
during tumor development. The GalCer treatment inhibits the subcutaneous tumor growth, tumor-induced
angiogenesis, and epithelial cell proliferation, which are required for tumor
vessel formation [57]. Later it has been established
that GalCer treatment, in combination with IL-21, prolonged and elevated the NK cell cytotoxicity, maturing NK cells into perforine-expressing cells by IL-21. Moreover, this combination inhibited spontaneous tumor metastases. Presentation of GalCer by DC to iNKT cells in contrast to soluble compound injection was even more effective in the suppression of metastasis formation [58]. Similar successful antitumor effects of GalCer-pulsed DC have been published by Toura et al. using B16 melanoma liver metastasis and lung metastasis of LLC model in vivo. Beside the inhibition of metastatic nodule formation in these tissues, GalCer-DC administration also has a significant beneficial effect in the regression of established nodules [59, 60].

The crosstalk between innate and adaptive immunity was established
in several anticancer studies. This linkage could be mediated via reciprocal interaction between iNKT cells and iDCs. Upon GalCer activation, iNKT cells express CD40L
[63]. The CD40 ligand binds to the CD40
molecules of DCs and triggers IL-12 expression and secretion by the DCs. The produced IL-12 generates a positive feedback and induces INF secretion by the iNKTs
[63, 64]. The secondary activation of DCs leads to NK, CD4+, and CD8+ T-cell activation by standard activation of memory T cells and adaptive immune response against peptides presented by DCs [65–67].

The fact that GalCer-loaded DC could trigger iNKT expansion and mediate antitumor
immune response in several in vitro experiments and in vivo
antimetastatic models in mice supported the notion that using GalCer-pulsed DCs for iNKT activation in cancer patients in situ might induce an effective anticancer
therapy. Other alternative approach could be the adoptive transfer of in vitro
expanded and activated iNKT cells to patients.

The number of NKT cells in cancer patients is significantly lower compared to
healthy volunteers. Giaccone et al. showed the disappearance of iNKT cells after 24 hours of GalCer administration from the peripheral blood of patients and only transient iNKT activation was
registered in some
individuals [68]. Others found quantitative defects in iNKT
cell-derived INF production among patients with advanced prostate cancer [69]. Showing that NK cells were able
to respond to IL-12,
those cells could secrete increased levels of INF demonstrating the selective loss of INF-secreting capacity of iNKT cells in patients [69]. As it was expected from mouse experiments, Chang et al. were able to expand the number of iNKT cells for more than a month in all treated patients, proving that GalCer-pulsed MDCs could be more effective than using GalCer-pulsed IDC or the soluble compound [70]. However, the levels of IL-12p40 and IL-10 in the serum were elevated after the treatment and iNKT cells showed reduced-INF secretion. The future challenge of this type of tumor therapy
is to induce extended iNKT number and activity. One possible approach is to use
additional pharmacological ligands upon cancer therapies. It has been shown that the
thalidomide analogue, lenalidomide (LEN), enhanced the predominant iNKT cell
expansion in vitro and in vivoin response to GalCer-loaded DC. LEN elicits higher level of INF secretion in response to GalCer-loaded DC, suggesting that LEN might be restoring the INF-producing activity of iNKT cells in cancer patients [71]. Alternative possibility is the adoptive transfer method: in phase I clinical trial, adoptive transferred iNKTs were used in patients with malignancy to increase the number of iNKT cells [72]. They expanded iNKT cells in vitro in
the presence of IL-2 and GalCer. The activated iNKT cells showed
cytotoxic activity against PC-13 and Daudi human cancer cell lines. Reinjection
of activated iNKT cell into the patients
enhanced the level of INF secreting iNKT cells in the peripheral blood from day one up to two weeks [72].

5. Dendritic Cell PPAR in Anticancer Therapy

Despite the enormous research effort,
cancer is still a significant clinical problem as well as basic science problem. However, immunotherapy opened up some possibility in the fight against cancer. General APC features of DCs are capturing antigens in the periphery, processing, and enhancing
MHC-peptide complex presentation capacity to naive T cells. These phenomena highlighted possible roles for DCs in anticancer therapy. However, DC-based vaccination often does
not elicit clinical responses and fails
to ensure long-time tumor regression in patients with malignant tumors. One reason for this failure could be the immunosuppressed state of the patient, for example, due to chemotherapy in the therapeutical history. Recently, we have identified a new
target gene, ABCG2, which is
transcriptionally regulated by PPAR in DCs. ABCG2 transporters modify the drug
resistance against anticancer
agent of PPAR agonist-treated DCs. PPAR has a protective function in these cells, and
using PPAR-specific ligands during in vitro differentiation could revert
the xenobiotics-induced
toxicity in DCs [73].

Due to the fact that we did not find reduced
capacity of DCs to activate T cell in MLR assays, we concluded that ligand activation does not suppress DC-mediated peptide antigen
presentation. For effective anticancer therapy, one should provoke adaptive
immune response against tumor-specific peptides presented by DCs. In mouse
experiments, simultaneously added GalCer and peptide-loaded
DCs induced CD4/CD8 T-cell-specific anticancer immune response mediated by iNKT cell. In case of human patients, GalCer-loaded DCs could
not induce adaptive immunity, partly because of the ineffective INF secretion by iNKT cells. Pharmacological
approaches like LEN may solve this problem. The ability of PPAR to upregulate CD1d expression on DCs raises the possibility to use receptor agonists in iNKT-based
adoptive transfer treatments.

Many features of DCs, which are critical during DC-vaccination
design, are affected by PPAR. Reduced migratory capacity, inhibited
IL-12 cytokine production, inadequate Th1 and CD8+ T-cell response, and presumed generation
of IL-10-producing tolerogenic DC could influence the outcome of DC-based vaccination
therapies against cancer. Based mainly on these in vitro results, activation of the PPAR receptor in tumor peptide-pulsed DCs could be less
beneficial in terms of in vivo
vaccination strategies. At
the same time, increased
phagocytic capacity, increased CD1d expression, and iNKT activation potential are useful features of PPAR-programed DCs. In spite of the vast amount of in vitro obtained results on the
potential role of PPAR in DCs, the most controversial issue remains open:
whether synthetic PPAR agonists have significant modifying effects on antitumor immune response in vivo or not. Further in vivo studies are needed
to clarify the receptor-specific immunomodulatory
effects of PPAR ligands (agonists or antagonists) in cancer
patients. For that, the use of siRNA-based gene-silencing techniques or DC-specific PPAR, KO animal models would probably be useful.

Acknowledgments

The work in the author’s laboratory
is supported by a grant from the National Research and Technology Office
RET-06/2004 to L. Nagy; and one from the Hungarian Scientific Research Fund
(OTKA no. NK72730). L. Nagy is an International Scholar of the Howard Hughes
Medical Institute and holds a Wellcome Trust Senior Research Fellowship in
Biomedical Sciences in Central Europe no. 074021.